This invention relates to ultrasonic vibration probes. More particularly, this invention relates to such an ultrasonic probe or horn assembly which is particularly useful in the simultaneous sonication of biological and cellular materials disposed in multiple wells of a tray.
It has been well known for decades that a probe which vibrates at ultrasonic frequencies (i.e. frequencies greater than 16,000 Hz) and has its distal end submerged under fluids will create cavitation bubbles if the amplitude of vibration is above a certain threshold. Many devices have been commercialized which take advantage of this phenomenon. An example of such an ultrasonic cellular disrupter is disclosed in the Sonicator(trademark) sales catalog of Misonix Incorporated of Farmingdale, N.Y. In general, devices of this type include an electronic generator for producing electrical signals with frequencies ranging from 16 to approximately 100 KHz, a piezoelectric or magnetostrictive transducer to convert the signal to mechanical vibrations and a probe (a.k.a. horn or velocity transformer) which amplifies the motion of the transducer to usable levels and projects or removes the operating face away from the transducer itself. The design and implementation of these components are well known to the art.
The cavitation bubbles produced by such ultrasonic vibration devices can be utilized to effect changes in the fluid or upon particles suspended therein. Such changes include biological cell disruption, deagglomeration of clumped particles, emulsification of immiscible liquids and removal of entrained or dissolved gases, among many others.
Cell disruption has been a particularly good application for probe type devices, in that the cells may be disrupted without the heat or cellular changes which prevent further analysis by conventional methodology. Many scientific protocols have been written which name the Sonicator(trademark) (or similar devices) as the instrument of choice for the procedure.
One characteristic of the probe type ultrasonic vibration devices which limit their use is the fact that the standard probes must be inserted directly into the fluid. Because the probe occupies volume as it is submersed, very small samples cannot be processed. In addition, the probe becomes contaminated with the fluid since the probe is in direct contact with the fluid. If the probe is subsequently dipped into another sample, contamination of that sample may occur. In some cases, this cross contamination renders the second sample unusable for analysis.
One way to mitigate these deficiencies is to have the probe tip separated from the sample by a membrane or other solid surface. If liquid is present on both sides of the membrane or surface, the acoustic waves will propagate through the membrane and transfer the cavitation forces to the second liquid volume without having the probe in direct contact with that second liquid volume. This membrane does not have to be elastic. In fact, experience shows that glass or hard plastic is an acceptable material. Consequently, glass and plastic test tubes and beakers are routinely used in this service. Misonix Inc. produces and sells a device called the Cup Horn(trademark) which uses this method of acoustic wave transfer to allow the researcher to segregate the probe from the sample.
One requirement for use of the Cup Horn is that the beaker or test tube diameter be significantly smaller than the distal diameter of the Cup Horn probe itself. This allows the acoustic energy to be relatively uniform across the diameter of the sample container. In addition, liquid is forced to surround the entire probe end in order to provide the transfer fluid for the acoustic wave. FIG. 1 shows the relationship of the Cup Horn probe 12, transfer fluid 14 and sample test tube. A cup 16 having a cylindrical sidewall 18, an inwardly extending annular flange 20 and a cylindrical sleeve 22 is mounted to the horn or probe 12 via a coupling sleeve 24 and a pair of O-rings 26 disposed in a region about a node of ultrasonic vibration of the probe. The transfer fluid not only covers a transverse end face 28 of probe 12, but also surrounds a substantial portion of the cylindrical distal surface 30 of the probe.
The requirements of (a) the relative sizes of the probe 12 and the test tube and (b) the surrounding of the probe end surface 30 by the transfer fluid 14 give rise to at least two problems. First, the size of the vessel is limited to that of the surface area of the probe 12 and second, the liquid 14 surrounding the probe 12 places a great load upon the probe. The power required to overcome this load is many times that needed for acoustic coupling into the small sample. In some cases, as the probe has been made larger to accommodate larger samples, the energy required has become greater than the power capability of the electronic generators currently available. In such cases, system overloads have occurred.
These limitations become especially apparent when the sample vessel takes the form of a multi-well microtiter plate or tray. Such a plate is typically made from clear hard plastic such as polystyrene, polyvinylchloride or acrylics. The tray is fairly shallow and may contain up to approximately 96 depressions (wells) into which the samples or specimens are placed. Each depression may contain only a few microliters of sample. In most cases, the insertion of a probe device is problematic since each sample must be isolated from the others, the wells are too small and the total processing time would be an unacceptable multiple of the processing time of one cell. Therefore, most researchers would prefer a device which would isolate the samples from the ultrasound probe and process all cells simultaneously.
It would be obvious to most persons skilled in the art to simply enlarge the diameter of the probe to allow the entire tray to be covered. However, as previously stated, the probe becomes very large, leading to non uniformity in the vibrational amplitude of the distal surface, very high power requirements and high cost of manufacture. In the past, probes of smaller square section were made which allow a quarter of the tray to be processed at a time, which decreased processing time substantially. However, most researchers required a further reduction in time in order to process their entire workload in one day. Also, the outer edges of the trays received irregular ultrasonic energy and therefore inconsistent cell breakdown in successive samples.
An object of the present invention is to provide an ultrasonic device which could treat a full microtiter tray simultaneously.
Another object of the present invention is to provide such an ultrasonic device which increases the degree of uniformity of acoustic intensity across the cells of the microtiter tray.
A further object of the present invention is to provide such an ultrasonic device which does not heat the fluid or the sample liquids, and which require minimum energy to operate, thereby allowing the use of the device on existing laboratory scale ultrasonic processors.
These and other objects of the present invention will be apparent from the drawings and descriptions herein.
The present invention is directed to an ultrasonic sonication device which includes two basic components, namely, (1) a velocity transformer (or probe) which, when coupled to a vibrating transducer of the piezoelectric or magnetostrictive type, resonates in sympathy with the transducer and either increases or decreases the magnitude of the transducer""s vibration and 2) a shallow cup assembly which holds a microtiter tray in a suitable orientation and contains an amount of liquid which provides efficient acoustic coupling.
An ultrasonic horn assembly comprises, in accordance with the present invention, an ultrasonic horn or probe having an axis and a distal end with an end face oriented substantially transversely to the axis. The end face of the probe is disposed at least approximately at an antinode of ultrasonic vibration of the horn or probe. A cup member is attached to the horn or probe at least approximately at the antinode so as to define a liquid reservoir covering the end face of the horn or probe. This attachment of the cup member at, or approximately at, the antinode at the distal end of the probe enables the formation of the reservoir as a shallow reservoir covering essentially only the end face of the probe. A small or marginal circumferential surface of the probe, contiguous with the end face thereof, may be submerged in the coupling liquid, as well.
In an ultrasonic horn assembly in accordance with the present invention, the load placed upon the probe is decreased owing to the reduction in the area of contact between the coupling fluid and the probe. The power requirements are accordingly reduced for a probe end face of a given area.
The cup member is attached to the horn or probe via a flexible coupling element such as an O-ring or an annular elastomeric membrane. Where the cup member includes a sidewall and a lower wall or flange extending inwardly from the sidewall, the lower wall is provided with at least one port for feeding liquid to the reservoir. Preferably, the port is one of at least a pair of ports disposed on substantially opposite sides of the cup member. The feeding of the coupling liquid through a lower wall of the cup member has advantages detailed below.
The end face of the probe is disposed in a first plane and an upper surface of the flange is disposed in a second plane spaced a first predetermined distance from the first plane, so that a lower surface of a specimen-containing tray resting on the upper surface of the flange is spaced a second predetermined distance from the probe end face. This spacing optimizes the acoustic effects of the ultrasonic energy on specimens contained in wells of a microtiter tray. To enable an optimal spacing, the probe end face is provided with a plurality of grooves for receiving peripheral lower edges of the tray so that contact between the tray and the vibrating probe is prevented.
Where the end face of the probe is circular, the end face has a diameter larger than a largest dimension of the portion of the tray containing the sample wells. Thus, all of the sample wells are located over the end face of the probe.
In accordance with another feature of the present invention, the probe is provided at the distal end, proximately to the end face, with an annular concavity for providing or enhancing uniformity of the ultrasonic wave field generated in the coupling fluid reservoir.
An ultrasonic sonication device in accordance with the present invention is an effective apparatus to acoustically treat or disrupt samples within a multiwell microtiter tray.